Mercury methylation by microorganisms: from cell to environment – MicroMer

The studies are conducted on three model sulfate-reducing bacteria, Pseudodesulfovibrio hydrargyri BerOc1, capable of methylating Hg(II) and demethylating MMHg, and on two strains capable only of demethylating MMHg, Pseudodesulfovibrio piezophilus C1TLV30 and Desulfovibrio alaskensis G20. Mutants of P. hydrargyri BerOc1 with deleted genes involved in the recognition, methylation, and export of Hg will be generated. The heterologous expression of these genes in D. alaskensis G20 and P. piezophilus C1TLV30 will also be carried out. Through adaptive evolution, we will produce hypermethylating and hyperdemethylating mutants of P. hydrargyri BerOc1. The impact on 1) methylation and demethylation, 2) speciation and ligands (thiols), and 3) the localization of Hg forms will be determined in these mutants.
MicroMer explores an original working approach, based on solid preliminary results, to decode Hg transformations. The coupling of genetic, bacterial physiology, imaging methods (nano X-ray fluorescence and electron microscopy), and X-ray absorption and mass spectroscopy approaches is unique and innovative; it will yield original results on Hg transformations. The MicroMer project also includes an environmental approach to determine to what extent the mechanisms described in bacterial models are widespread. Through metagenomic and metatranscriptomic methods conducted on sediments from the Etang de Berre, where P. hydrargyri BerOc1 was isolated, we will assess the representativeness of the identified cellular Hg transformation mechanism. In parallel, we will determine the diversity and expression of genomic markers involved in Hg transformation, as well as their dynamics in daily and seasonal cycles.

The speciation of Hg is currently considered a key factor determining the availability of Hg and, consequently, the net production of MMHg by microorganisms. The study of mutants of P. hydrargyri BerOc1 incapable of methylating Hg(II) showed different speciation of Hg(II) compared to the wild-type strain, suggesting that the cell modifies the speciation of Hg(II) during the methylation process. Other mutants of P. hydrargyri BerOc1, deleted of several genes involved in resistance, sensing, or metal efflux, showed that the accumulation of Hg(II) inside the cell is affected, but not that of MMHg. Furthermore, the transformations of Hg (methylation of Hg(II) or demethylation of MMHg) are altered. These results indicate a coupling of the speciation of Hg(II), intracellular accumulation, and Hg transformations by the cells. These results will ultimately enable the development of a model for Hg detection by the cell, speciation in the cellular environment and inside the cell, Hg transformation at the intracellular level, and export of different forms of mercury. We have obtained strains adapted to high concentrations of mercury (up to 40 µM of Hg(II)), and their physiological and molecular study is underway. We have also heterologously expressed the hgcAB genes in a non-methylating sulfate-reducing strain and obtained Hg(II) methylation activity. These different strains should strengthen the model generated regarding the link between Hg speciation, cell physiology, genomics, genetics, and methylmercury production.
The model generated is further supported by the fact that the model strain used in this study (P. hydrargyri BerOc1) is consistently found during various sampling campaigns in its original environment (Etang de Berre, on the French Mediterranean coast), highlighting the representativeness of this model strain in the environment. More significantly, during certain campaigns, the population associated with this strain clearly dominated the community of microorganisms capable of Hg(II) methylation (Vigneron et al., 2021)

The work continues with experiments aiming to generate other mutants adapted to high concentrations of MMHg, as well as mutants of P. hydrargyri BerOc1 deleted of other targeted genes. The physiological, genomic, and genetic characterization of these strains will improve the current model. Some of the genes selected during the study of different mutant strains of P. hydrargyri BerOc1 will then be used to perform heterologous expression in sulfate-reducing strains incapable of producing MMHg. Imaging approaches will complement the data already acquired on the localization and speciation of mercury.

Peer reviewed publications
- Vigneron A, Cruad P, Aubé J, Guyoneaud R, Goñi-Urriza M. 2021. Transcriptomic evidence for versatile metabolic activities of mercury cycling microorganisms in brackish microbial mats. npj Biofilms and Microbiomes, 7, 83 DOI: 10.1038/s41522-021-00255-y
- Bakour I, Isaure MP, Barrouilhet S, Goñi-Urruza M, Monperrus M. 2023. Coupling fluorescent probes to characterize S-containing compounds in a sulfate reducing bacteria involved in Hg methylation. Talanta, 7, 100228
- Barrouilhet S, Monperrus M, Tessier E, Khalfaoui-Hassani B, Guyoneaud, Isaure MP, Goñi-Urriza M. 2023. Effect of exogenous and endogenous sulfide on the production and the export of methylmercury by sulfate reducing bacteria. Environmental Science and Pollution Research, 30, 3835-3846.


Conferences presentations
- Barrouilhet S., Isaure M-P, Dolla A., Gassie C., Khalfaoui-Hassani B., Guyoneaud R., Monperrus M., Goñi Urriza M. (2023) First evidence of a mercury resistance mechanism in an anaerobic bacterium: impact on mercury sensitivity, accumulation and, methylation. Goldschmidt2023, 9-14 July. Oral
- Barrouilhet S., Isaure M-P, Dolla A., Gassie C., Le Bars M., Khalfaoui-Hassani B., Guyoneaud R., Monperrus M., Goñi Urriza M. (2022). Identification d’un premier mécanisme de résistance au mercure chez les bactéries anaérobies : mise en évidence d’une autre voie que l’opéron mer. 17th Conference of the Société Française de Microbiologie, 3-5 October 2022, Montpellier, France. Poster
- Le Bars M., Barrouilhet S., Monperrus M., Goñi Urriza M., Rovezzi R., Salomé M., Isaure M-P. (2022). Mercury methylation and transformations by the sulfate reducing bacterium Pseudodesulfovibrio hydrargyri combining synchrotron cryo-nano-XRF, XRF tomography and HERFD-XANES. 30th Goldschmidt Conference, 10-15 July 2022, Honolulu, Hawaï, USA (on site and virtual). Oral
- Bakour I., Barrouilhet S., Goñi Urriza M., Isaure M-P, Monperrus M. (2022). Coupling fluorescent probes to characterize S-containing compounds in a mercury methylating sulfate reducing bacteria. 15th International Conference on Mercury as a Global Pollutant, 24-29 July 2022, Cape Town, South Africa (Virtual). Oral
- Bakour I., Isaure M-P, Barrouilhet S., Goñi Urriza M., Monperrus M. (2023) Impact of Cysteine on Hg Methylation and Demethylation in the Sulfate Reducing Bacterium Pseudodesulfovibrio hydrargyri BerOc1. ICOBTE/ICHMET22. ICOBTE/ICHMET23 6-10 September. Wuppertal, Germany. Oral

Mercury (Hg) is a global pollutant, able to be converted into highly neurotoxic monomethylmercury (MMHg), a compound bioaccumulated and bioamplified in food webs. Microorganisms regulate environmental MMHg level, by controlling directly inorganic Hg (IHg) methylation and MMHg degradation or indirectly through redox transformations controlling Hg bioavailability. Understanding the biotransformation processes of Hg in the environment is a key component of risk assessment of Hg in ecosystems and human health. However, there is little knowledge on the cellular processes leading to MMHg production. It is of special interest to develop studies at a cellular level to understand Hg transformations in terms of genetic determinism, cellular pathways and environmental factors regulating them.

The MicroMer project aims to characterize the process of Hg methylation and demethylation at cellular level and environmental level. At cellular level, MicroMer aims to determine 1) the speciation of Hg in the cell environment favoring Hg transformations, 2) the mechanisms of Hg recognition by the cell, 3) the intracellular steps of Hg speciation and, 4) the Hg species export from the cell. Our hypothesis is that methylation and demethylation processes are coupled and that they are driven by Hg uptake but also its export. Thus, we intend to decipher the role of Hg cell trafficking and Hg speciation (in the extracellular and intracellular compartments) in Hg transformations. The processes will be investigated in Sulfate Reducing Bacteria models, Pseudodesulfovibrio hydrargyri BerOc1, able to methylate IHg and demethylate MMHg and two other strains able only to demethylate MMHg: Pseudodesulfovibrio piezophilus C1TLV30 and Desulfovibrio alaskensis G20. P. hydrargyri BercOc1 mutants of either Hg methylation, sensing, and export, and their heterologous expression in C1TLV30 and G20 will be performed. By experimental evolution, we will also generate BerOc1 strains with higher methylation and demethylation capacities. The consequences of mutations in 1) Hg methylation and demethylation, 2) Hg speciation and nature of Hg ligands (thiols), and 3) localization will be determined. MicroMer explores an outstanding and original line of work to understand Hg transformation based on our solid previous results. The striking combination of genetics, bacterial physiology and imaging methods (nano X-ray fluorescence, and electron microscopy) coupled with X-ray absorption spectroscopy and hyphenated mass spectrometry techniques is, to our knowledge, unique and innovative, and will forcefully bring new results and perspectives in the understanding of Hg methylation and demethylation. The MicroMer project has also an environmental scope that aims to determine the representativeness of the mechanism described in our model strains. By applying metagenomics and metatranscriptomics in a time-dependent (daily and seasonally) approaches, we will determine the fate of our model strain in its original environment. In parallel, we will determine the diversity and expression of major genetic determinisms in order to gain an overview of the representativeness of the model deciphered in MicroMer project in a natural environment.

The MicroMer project proposes extensive and collaborative studies on mercury transformations mediated by bacteria using interdisciplinary approaches (molecular genetics, microbial physiology, analytical chemistry, X-ray absorption spectroscopy, imaging and microbial ecology). The project unites research teams (IPREM, MIO, and BIC) with strong expertise on those approaches in order to shed light on challenging topic. The environmental and health implications of the expected data obtained in this basic research project are of major interest. They will provide key information about the highly toxic MMHg production that is essential for risk evaluation, management, and sustainable development.